Protective Effect And Mechanism Of Swertia Cincta Burkill On Acute Liver Failure Induced By LPS/D-GalN In Mice

Acute liver failure(ALF)is a clinical syndrome with a high mortality rate.Common etiological factors include viral infections,liver ischemia-reperfusion injury,drug overdose,specific drug reactions,and toxic substances.Currently,liver transplantation is the only effective treatment for ALF.However,due to its high cost and limited availability of donor livers,there is an urgent need to find new effective drug treatments for ALF.Swertia cincta,widely distributed in southwestern China,is extensively used in traditional medicine for the treatment of hepatitis and other liver diseases.However,its protective effects and mechanisms against ALF have not been studied.In our previous experimental studies,the ethanol extract of Swertia cincta(ESC)demonstrated significant hepatoprotective effects.Therefore,this study aims to investigate the protective effects of ESC on a mouse model of acute liver failure induced by lipopolysaccharide(LPS)in combination with Dgalactosamine-2-n-sulfate(D-Gal N),and elucidate its underlying mechanisms.The study provides a theoretical basis for the application of ESC.The main research contents and results are as follows:1 Identification and screening of active components in ESCThe active components in ESC were detected using liquid chromatography-tandem mass spectrometry(LC-MS),followed by screening the identified active components based on the principles of drug likeness and their bioavailability score.Furthermore,the toxicological properties of the active components in ESC were predicted using the Pro Tox II database.Results:(1)After LC-MS analysis of ESC,a total of 41 potential components were initially identified.After screening,32 main active components were retained for subsequent network pharmacological analysis.(2)The major active components in ESC belong to the categories of flavonoids,cyclic terpenes,oxygenated anthraquinones,and terpenoids.(3)The major active components in ESC were found to be generally non-toxic or low in toxicity.Conclusion: ESC primarily contains flavonoids,cyclic terpenes,oxygenated anthraquinones,and terpenoids,and does not exhibit additional toxicity to the liver.2 Network pharmacologyAfterward,the Swiss Targets Prediction and Genecard databases were utilized to predict the targets of ESC and acute liver failure(ALF)separately,and the intersection of the two sets of targets was determined.The Cytoscape software was then employed to predict the Degree values of the intersected targets,and the top ten targets based on Degree values were considered as core targets.The possible mechanisms of ESC in treating ALF were predicted using the GO and KEGG databases.Subsequently,molecular docking and molecular dynamics simulations were performed to preliminarily validate the core targets,followed by further validation using the GEO database.Results:(1)A total of 383 drug targets and 547 disease targets were identified,with 72 intersected targets.The top ten targets based on Degree values were ALB,ERBB2,AKT1,MMP9,EGFR,PTPRC,MTOR,ESR1,VEGFA,and HIF1 A.(2)Based on the enrichment of targets by the active components of ESC,key components in ESC,such as santalin,musizin,were determined.(3)The most significant signaling pathways identified by KEGG enrichment analysis were the EGFR signaling pathway and PI3K/AKT signaling pathway.(4)Among the ten core targets,eight targets showed differential expression in ALF patients.PTPRC,MMP9,and HIF1 A were significantly upregulated in ALF patients,while VEGFA,ALB,AKT1,EGFR,and ESR1 were significantly downregulated.(5)Molecular docking and molecular dynamics simulation results showed that Eriodictyol,kaempferol,and luteolin exhibited the best docking scores with EGFR,indicating stable binding.Conclusion: ESC may exert its therapeutic effects on ALF by modulating targets such as ALB,ERBB2,AKT1,MMP9,EGFR,PTPRC,MTOR,ESR1,VEGFA,and HIF1 A,as well as the EGFR and PI3K-AKT signaling pathways.Eriodictyol,kaempferol,and luteolin are likely the main active components in ESC responsible for their pharmacological effects.3 Protective effect of ESC on LPS/D-Gal N-induced ALF miceMice were randomly divided into seven groups: blank control group,model group,positive drug group,ESC control group,and low,medium,and high dose ESC groups.Corresponding treatments were administered to the mice for 7 days.On the 7th day,except for the blank control group and ESC control group,ALF was induced in the other groups by intraperitoneal injection of LPS/D-Gal N.After 6 hours of modeling,samples were collected.Serum biochemical markers,oxidative stress indicators,and inflammatory factors were measured,and liver tissue was subjected to HE staining and TUNEL staining to investigate the effects of ESC on liver function,liver tissue structure,oxidative stress,inflammatory levels,and hepatocyte apoptosis in ALF mice.Results:(1)ESC reduced the levels of ALT,AST,TP,and ALB in the serum of ALF mice,alleviating liver function damage.(2)ESC increased the levels of CAT,SOD,and GSH in the serum of ALF mice and decreased the accumulation of MDA,thus reducing liver oxidative damage.(3)ESC pretreatment significantly reduced the liver index of ALF mice and improved liver tissue pathological changes.(4)ESC decreased the levels of IL-6,TNF-α,and IL-1β in the serum of ALF mice,thereby alleviating the inflammatory response.(5)ESC reduced the number of apoptotic cells in the liver of ALF mice and alleviated the level of apoptosis.Conclusion: ESC exerts a protective effect on ALF mice mainly through its anti-apoptotic and antioxidant properties.4 Protective effect of ESC on LPS/D-Gal N-induced Hep G2 cell injuryBased on the in vivo experimental results,further investigations were conducted at the cellular level.The cell viability of Hep G2 cells treated with different concentrations of ESC and D-Gal N was determined using the CCK-8 assay to establish the modeling concentration and safe concentration of ESC.After grouping,PI/AM fluorescence staining,ROS fluorescence staining,JC-10 fluorescence staining,flow cytometry apoptosis detection,biochemical markers,and cytokine measurements were performed to study the effects of ESC on cell function,apoptosis level,mitochondrial membrane potential,inflammatory levels,and oxidative damage in Hep G2 cells.Results:(1)When the concentration of D-Gal N was 30 m M,it significantly affected the cell viability of Hep G2 cells,with cell viability below 50%.Therefore,LPS at 1 μg/ml in combination with DGal N at 30 m M was used as the modeling concentration.When the concentration of ESC was less than 50 μg/ml,it did not significantly affect the cell viability of Hep G2 cells.Therefore,the concentration of ESC should be controlled within 50 μg/ml,with dosages of25,12.5,and 6.25 μg/ml used for treatment.(2)ESC significantly reduced the levels of ALT and AST in the culture medium of Hep G2 cells,and also decreased the levels of IL-6,TNF-α,and IL-1β inside the cells,thereby alleviating the inflammatory response.(3)ESC significantly reduced the cell apoptosis in LPS/D-Gal N-induced Hep G2 cells.(4)ESC significantly decreased the levels of reactive oxygen species(ROS)in Hep G2 cells and reversed the decrease in mitochondrial membrane potential induced by LPS/D-Gal N,thus improving oxidative damage in Hep G2 cells.Conclusion: ESC alleviates LPS/D-Gal Ninduced Hep G2 cell injury mainly by improving apoptosis and oxidative damage.5 Mechanism of the hepatoprotective effect of ESCValidation of the core target genes predicted by network pharmacology for the treatment of ALF by ESC was performed using q PCR.Additionally,Western blot analysis was conducted to measure the expression levels of p-AKT,p-PI3 K,p-m TOR,p-EGFR,and p-ERK1/2,which are related proteins of the EGFR and PI3K/AKT pathways predicted by network pharmacology.Considering the close relationship between ESC and antioxidative damage in exerting hepatoprotective effects,the effects of ESC on proteins associated with the Nrf2-HO-1 signaling pathway,including HO-1,intracellular Nrf2,and extracellular Nrf2,were further examined.Results: ESC upregulated the expression levels of p-AKT,p-PI3 K,p-m TOR,HO-1,and intracellular Nrf2,while downregulating the expression levels of p-EGFR,p-ERK1/2,and extracellular Nrf2.ESC was found to exert anti-apoptotic effects by activating the EGFR and PI3K/AKT pathways and exerted antioxidative damage by promoting nuclear translocation of Nrf2 and activating the Nrf2 signaling pathway.In summary,ESC effectively alleviates liver pathology,oxidative stress,inflammation,and hepatocyte apoptosis in ALF mice,ultimately providing hepatoprotection.In in vitro experiments,ESC mitigates oxidative damage,mitochondrial dysfunction,and cell apoptosis in LPS/D-Gal N-induced Hep G2 cells,thereby enhancing cell viability and exerting a protective effect on the cells.The underlying mechanism may involve the modulation of core target genes(EGFR,HIF1 A,VEGFA,AKT1,ESR1,ERBB2,and m TOR)and related signaling pathways(EGFR/ERK,PI3K/AKT,and Nrf2/HO-1)to achieve antioxidative and anti-apoptotic effects,thus exerting hepatoprotection.